Project Summary Helicases are molecular motor proteins that use energy from hydrolysis of ATP to manipulate DNA and RNA in all phases of nucleic acid metabolism. Numerous mutations have been identified in many different helicases that are associated with human diseases including cancer, heart disease, and neurological disorders. The primary function of helicases is to unwind duplex DNA, but other critical functions have been discovered for which biochemical mechanisms are unknown. Helicases displace proteins from DNA and unfold secondary structures in DNA such as G-quadruplex DNA (G4DNA) in reactions that are critical for maintaining genomic stability. G4DNA is made of four guanines that form Hoogsteen hydrogen bonds in a planar ring which is referred to as a G quartet. Multiple stacks of these G quartets associate to form highly stable structures. G4DNA affects DNA metabolism including transcription, recombination, and replication. The Pif1 family of helicases has been identified in all eukaryotes and has been identified as playing a key role in recognition and unfolding of G4DNA structures. Mutation in Pif1 can increase the risk for some forms of breast cancer. The overall goals of this project are to determine the mechanism(s) by which Pif1 and other helicases push proteins from DNA and unfold critical DNA structures such as G4DNA. We will determine how helicases are affected by proteins with which they interact such as single-stranded binding proteins and recombinases. G4DNA sequences are found throughout the genome, but are localized preferentially to certain regions such as promoters of proto-oncogenes, telomeres, and mitochondrial DNA. The mechanism(s) through which these structures impart biological function are largely unknown. We have devised a method to examine the epiproteome at practically any site in the genome by using a CRISPR-Cas9 targeting strategy. We will identify the proteins and histone modifications that surround G4DNA sites in order to understand how these sequences influence gene expression, recombination, and other activities. We have applied a proteomic screen to discover new proteins that bind to G4DNA. The major proteins identified, including the RNA helicase DHX36, are known to assemble into cytoplasmic structures termed stress granules under conditions of cellular stress. The location of these proteins and their known roles in regulation of translation led us to test a hypothesis for one function of G4DNA. Our data supports the conclusion that G4DNA is excised from damaged mitochondrial and nuclear genomes and can enter the cytoplasm intact where it facilitates formation of stress granules. Our goals now are to determine the specific sequences of G4DNA removed from the genome, the mechanism by which the G4DNA is excised, and the specific functions by which excised G4DNA affects translation. The long-term goal is to understand how signaling by G4DNA overlaps and intersects with other signaling pathways such as the DNA damage response and innate immune response.